Lowry Protein Assay

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Questions and Answers

Considering the Lowry assay's sensitivity to pH, which buffer system would be most appropriate for preparing protein samples to ensure accurate quantification?

  • A carbonate-bicarbonate buffer system, meticulously adjusted to a narrowly controlled alkaline pH of 10.25 ± 0.05, validated with a high-resolution pH meter. (correct)
  • A Tris-HCl buffer at pH 7.0, rigorously maintained and frequently recalibrated.
  • A phosphate buffer at its pKa, supplemented with protease inhibitors adjusted to pH 10.0-10.5 using concentrated NaOH.
  • A buffer system employing zwitterionic compounds, carefully titrated to a pH range of 6.0-8.0 and further verified by mass spectrometry.

In the Lowry assay, the Folin-Ciocalteu reagent reacts primarily with which amino acid residues to produce a detectable chromophore?

  • Proline and hydroxyproline, leading to a unique spectral shift easily differentiated from tyrosine and tryptophan.
  • Tyrosine and tryptophan residues, engaging in redox reactions that reduce the phosphomolybdic-phosphotungstic heteropoly acids to heteropoly blue species. (correct)
  • Cysteine and methionine residues, resulting in the formation of disulfide bonds and subsequent detection using Ellman’s reagent.
  • Lysine and arginine residues, forming stable Schiff bases detected at 540 nm.

Given the potential interference of various substances in the Lowry assay, which pre-treatment strategy would most effectively mitigate the impact of lipid contamination in a complex biological sample?

  • Implementing a rigorous dialysis step using a membrane with a molecular weight cut-off specifically optimized to retain proteins while allowing lipids to diffuse out.
  • Performing a chloroform-methanol extraction to separate lipids from proteins, followed by reconstitution of the protein fraction in a compatible buffer. (correct)
  • Treating the sample with a high concentration of chaotropic agents like urea and thiourea to solubilize and mask the interfering effects of lipids.
  • Precipitating proteins with trichloroacetic acid (TCA) followed by multiple washes with ice-cold acetone to remove lipids.

When performing the Lowry assay, why is the order of reagent addition, particularly the sequence of alkaline copper reagent and Folin-Ciocalteu reagent, critically important for achieving accurate and reproducible results?

<p>The alkaline copper reagent must be added first to facilitate the complexation of copper ions with peptide bonds, which is essential for subsequent reduction of the Folin-Ciocalteu reagent. (A)</p> Signup and view all the answers

If the absorbance values obtained from a Lowry assay consistently exceed the upper limit of the standard curve, which of the following adjustments to the procedure would be most appropriate to ensure accurate protein quantification?

<p>Dilute the protein samples to fall within the linear range of the standard curve, while ensuring the dilution factor is accurately accounted for in the final calculation. (B)</p> Signup and view all the answers

Considering the Beer-Lambert Law and its application in spectrophotometric protein quantification, which modification to the Lowry assay would most effectively enhance the assay's sensitivity for detecting very low protein concentrations?

<p>Employing a cuvette with an extended path length to increase the effective absorbance of the sample. (B)</p> Signup and view all the answers

Given the variability in amino acid composition across different proteins, which normalization strategy would most accurately address the inherent limitations of using Bovine Serum Albumin (BSA) as a universal standard in the Lowry assay?

<p>Generating a protein-specific standard curve using a purified sample of the target protein, quantified via amino acid analysis or quantitative amino acid sequencing. (B)</p> Signup and view all the answers

When troubleshooting inconsistencies in Lowry assay results across multiple experiments, what specific control experiment would best differentiate between issues arising from reagent degradation versus procedural variations?

<p>Running a series of dilutions of a known BSA standard using freshly prepared reagents alongside the aged reagents, comparing the resulting standard curves. (D)</p> Signup and view all the answers

In the context of high-throughput protein quantification, what modification to the standard Lowry assay protocol would be most effective in minimizing reagent consumption while maintaining assay accuracy and reproducibility?

<p>Scaling down the reaction volumes to a microplate format, optimizing reagent concentrations, and employing a sensitive microplate reader. (B)</p> Signup and view all the answers

Considering the chemical mechanisms underlying the Lowry assay, which modification would likely improve its tolerance to high concentrations of reducing agents such as Dithiothreitol (DTT) commonly used in protein biochemistry?

<p>Incorporating a DTT-specific quenching step involving a thiol-reactive compound that selectively inactivates DTT without interfering with protein quantification. (B)</p> Signup and view all the answers

What is the primary reason Bovine Serum Albumin (BSA) is universally used as a standard protein in most protein estimation techniques?

<p>Because of its low cost, high purity, ready availability, and stability in various solutions. (B)</p> Signup and view all the answers

In the Lowry method, what is the purpose of the copper ions in the alkaline copper reagent?

<p>To bind to the peptide bonds of the protein, facilitating the subsequent reduction of the Folin-Ciocalteu reagent. (C)</p> Signup and view all the answers

What is the wavelength at which the blue color developed in the Lowry assay is typically measured?

<p>660 nm (A)</p> Signup and view all the answers

Certain compounds can interfere with the Lowry assay. Which of the following is NOT listed as an interfering substance?

<p>Glycerol (A)</p> Signup and view all the answers

In preparing the alkaline copper reagent (Reagent C), what is the ratio of Reagent A to Reagent B?

<p>50:1 (A)</p> Signup and view all the answers

During the Lowry assay procedure, after adding the alkaline copper reagent, how long should the mixture stand at room temperature before adding the Folin-Ciocalteu reagent?

<p>10 minutes (A)</p> Signup and view all the answers

After adding the Folin-Ciocalteu reagent, how long should the mixture incubate in the dark at room temperature?

<p>30 minutes (A)</p> Signup and view all the answers

How is the 'blank' prepared in the Lowry assay procedure?

<p>By using a tube with distilled water only. (B)</p> Signup and view all the answers

What is the concentration of the 'working standard' of BSA after dilution as described in the provided procedure?

<p>200 µg/ml (C)</p> Signup and view all the answers

The principle of Lowry's method relies on the reaction between the Folin-Ciocalteu reagent and which components?

<p>Aromatic amino acids (A)</p> Signup and view all the answers

A researcher observes a greenish color in their Folin-Ciocalteu reagent. What does this indicate?

<p>The reagent should not have any greenish color, and this suggests it's unsuitable for use (A)</p> Signup and view all the answers

The intensity of the color produced in the Lowry method is directly related to what?

<p>The amount of aromatic amino acids present (C)</p> Signup and view all the answers

What is the purpose of boiling the mixture with lithium sulfate and bromine water during the alternate preparation of Folin-Ciocalteu reagent?

<p>To remove excess bromine (B)</p> Signup and view all the answers

The Lowry method, although widely used, has a limitation in that the exact mechanism of color formation is...

<p>Poorly understood (C)</p> Signup and view all the answers

To ensure the most accurate results when using the Lowry method, what should one do regarding interfering substances?

<p>Remove or dilute them to levels where their effect is insignificant. (C)</p> Signup and view all the answers

Flashcards

Aim of Lowry's Method

To estimate the total protein amount in a sample using Lowry's method.

Principle of Lowry's Method

Phenolic groups of tyrosine and tryptophan in a protein react to produce a blue-purple complex, measured at 660 nm.

Components of Folin-Ciocalteau Reagent

Sodium tungstate, molybdate, and phosphate.

Standard Protein in Lowry Assay

Bovine Serum Albumin, due to its low cost, high purity, and ready availability.

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Interfering Compounds in Lowry Assay

Tris, zwitterionic buffers, EDTA, carbohydrates, lipids, sulfhydryl reagents, thiols, and some salts.

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pH Sensitivity of Lowry Method

Maintain between 10 - 10.5 for accurate results.

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Lowry's Reagent A

2% Na2CO3 in 0.1 N Sodium hydroxide

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Lowry's Reagent B

0.5% CuSO4 in 1% Sodium potassium tartrate

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Lowry's Alkaline Copper Reagent (Reagent C)

Mix 50 ml of reagent A and 1ml of reagent B prior to use

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Folin-Ciocalteau reagent (FC)

This reagent is available commercially in a form ready for use.

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Preparation of Folin-Ciocalteau reagent

Reflux gently for 10 hours with a mixture consisting of 100g of sodium tungstate (Na2WO4.2H2O), and 25 g of sodium molybdate, (Na2MoO4.2H2O), 70 ml of water, 50 ml of 85% phosphoric acid, 100 ml of conc. HCl in a 1.5 litre flask. Add 150g lithium sulphate (Li2SO4.H2O), 50ml of water and a few drops of bromine water. Boil the mixture for 15 minutes without condenser to remove excess bromine. Allow to cool, dilute to 1 litre with distilled water and filter. The reagent should have no greenish colour.

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Protein Standard Stock

Weigh 50 mg of BSA (fraction 5) and dissolve in distilled water and make up 50 ml (1mg/ml).

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Working Standard

Dilute 10ml of standard stock to 50 ml with distilled water (200µg/ml).

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Volume of working standard

Use 0.2, 0.4, 0.6, 0.8, and 1ml of BSA.

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Wavelength measured at

Measure the blue colour at 660nm.

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Study Notes

  • Lowry's method is used to estimate the total protein in a sample

Principle of Lowry's Method

  • Tyrosine and tryptophan residues in a protein react to produce a blue-purple complex.
  • Folin-Ciocalteau reagent (FC), containing sodium tungstate, molybdate, and phosphate, is used in the reaction.
  • Maximum absorption occurs at 660 nm.
  • Color intensity depends on the amount of aromatic amino acids present.
  • Bovine Serum Albumin (BSA) is commonly used as a standard protein due to its low cost, high purity, and availability.
  • The method is sensitive to about 10 g/ml.
  • The exact mechanism of color formation is poorly understood.
  • Interference can occur due to Tris, zwitter ionic buffers, PIPES, HEPES, EDTA, carbohydrates, lipids, sulfhydryl reagents, thiol compounds, and some salts.
  • Interfering substances should be removed or diluted.
  • The Lowry method is sensitive to pH changes, so the pH of the assay solution should be maintained at 10 - 10.5.
  • Incubation time is critical for reproducible results.

Materials and Methods

  • Reagent A: 2% Na2CO3 in 0.1 N Sodium hydroxide.
  • Reagent B: 0.5% CuSO4 in 1% Sodium potassium tartrate.
  • Reagent C: Alkaline Copper reagent (mix 50 ml of reagent A and 1 ml of reagent B).
  • Follin-Ciocalteau reagent (FC) is available commercially ready for use.
  • FC reagent can be prepared by refluxing a mixture of sodium tungstate (100g), sodium molybdate (25g), water (70 ml), 85% phosphoric acid (50 ml), conc. HCl (100 ml) in a 1.5-litre flask for 10 hours. Add lithium sulphate (150g), water (50ml) and bromine water, boil for 15 minutes, cool, dilute to 1 litre, and filter (Reagent D).
  • Protein standard stock: Weigh 50 mg of BSA (fraction 5), dissolve in distilled water, and make up to 50 ml (1mg/ml).
  • Working standard: Dilute 10ml of standard stock to 50 ml with distilled water (200g/ml).

Procedure

  • Pipette 0.2, 0.4, 0.6, 0.8, and 1ml of the working standard (BSA) into test tubes.
  • Pipette 0.1 ml and 0.2 ml of the unknown sample into test tubes.
  • Adjust the volume to 1ml in all test tubes using distilled water, a tube with water only is the blank.
  • Add 5ml of reagent C to all test tubes and rapidly mix and stand for 10 minutes.
  • Add 0.5 ml of reagent D (FC), mix, and incubate at room temperature in the dark for 30 minutes.
  • The blue color is measured at 660nm.
  • The amount of protein in the sample is calculated by creating a standard graph.

Table reagents

Reagents BLK 1 2 3 4 5 UK1 UK2
BSA standard (ml) - 0.2 0.4 0.6 0.8 1.0 0.1 0.2
BSA concentration (μg) - 40 80 120 160 200 ? ?
Volume of distilled water (ml) 1.0 0.8 0.6 0.4 0.2 - 0.9 0.8
Alkaline copper reagent (ml) 5 5 5 5 5 5 5 5
FC reagent (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
  • Stand at room temperature for 10 minutes
  • Incubate at room temperature in the dark for 30 minutes
  • Measure Absorbance at 660nm

Reference

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent J. Biol. Chem. 1951, 193, 265-275.

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